Creatine kinase is an essential piece of the contractile apparatus. In themyocardium, inhibition of creatine kinase leads to contractile dysfunction. As creatine kinase contains a reduced cysteine residue that is essential for substrate binding, it follows that this enzyme may be susceptible to oxidative damage from, for example, the oxidative stress associated with ischemia/reperfusion injury. The objective of this study was to examine the effects of nitric oxide and its metabolites on the activity of creatine kinase. Enzyme activity was measured by following NADH oxidation in a coupled assay system. Peroxynitrite was synthesized from hydrogen peroxide and S-nitrosoglutathione (GSNO) was synthesized from gluthathione. Nitric oxide and superoxide were simultaneously generated using either SIN-1 or a combination of spermine NONOate and xanthine/xanthine oxidase. Nitric oxide, GSNO and peroxynitrite all inhibit creatine creatine kinase activity with relative potency p erox ynitrite>>GSNO>NO. Full activity could be restored to enzyme inhibited with either GSNO or NO by incubation with thiol reducing agents (GSH or dithiothreitol). In contrast only 5-20% of enzyme activity could be recovered from enzyme inhibited with peroxynitrite, SIN-1 or a combination of spermine NONOate and xanthine/xanthine oxidase. In order to determine if thiol oxidation is an essential component of peroxynitrite-dependent inhibition, the thiol groups of creatine kinase were protected by pre-treatment of the enzyme with a high concentration of GSSG. Addition of an inhibitory concentration of peroxynitrite to this preparation, followed by incubation with GSH resulted in regeneration of full activity. This strongly suggests that thiol oxidation is an essential component of inhibition. The rate constant for the reaction between peroxynitrite and creatine kinase was determined to be 8.84 x 10(5) M(-1)s(-1) by competition analysis. In all experiments, activity and the concentration of p rotein thiols were well correlated. Decomposed peroxynitrite had no effect on creatine kinase or upon the creatine kinase assay. These data indicate that nitric oxide and GSNO inhibit CK by oxidizing an essential thiol residue to either a sulfonic acid, a mixed disulfide or a protein S-nitrosothiol, all of which can be repaired by exogenous thiol. However, peroxynitrite oxidizes the active site thiol to a form that cannot be reduced by additional thiol. The measured rate constant is doubt 10(3) times greater than the reaction between peroxynitrite and GSH, perhaps reflecting the high positive charge density in the active site of CK.